Coexpression of Ligand-gated P2X and G Protein-coupled P2Y Receptors in Smooth Muscle

Abstract

P 2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca 2؉ release, and contraction that were abolished by U-73122 and guanosine 5-O-(3-thio)diphosphate, and partly inhibited (50 -60%) by pertussis toxin (PTX). ATP analogs (adenosine 5-(␣,␤-methylene)-triphosphate, adenosine 5-(␤,␥-methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca 2؉ influx and contraction that were abolished by nifedipine and in Ca 2؉ -free medium. Micromolar concentrations of ATP stimulated both Ca 2؉ influx and Ca 2؉ release. ATP and UTP activated G q/11 and G i3 in gastric and aortic smooth muscle and heart membranes, G q/11 and G i1 and/or G i2 in liver membranes, and G o and G i1-3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by G␣ q/11 -dependent activation of phospholipase (PL) C-␤1 and G␤␥ i3 -dependent activation of PLC-␤3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to G␣ q/11 , G ␤ , PLC-␤1, or PLC-␤3, and completely inhibited by the following combinations (PLC-␤1 and PLC-␤3 antibodies; G␣ q/11 and G ␤ antibodies; PLC-␤1 and G ␤ antibodies; PTX with either PLC-␤1 or G␣ q/11 antibody).The pattern of responses implied that P 2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-␤1 via G␣ q/11 and to PLC-␤3 via G␤␥ i3 . These receptors co-exist with ligand-gated P 2X1 receptors activated by ATP analogs and high levels of ATP. P 2 receptors have been classified recently into two classes comprising ligand-gated cationic channels or P 2X receptors and G protein-coupled P 2Y receptors (1, 2); P 2U and P 2T receptors have been subsumed into the P 2Y class of receptors. The term P 2 recognizes the fact that purine and pyrimidine nucleotides can act as preferential ligands of various receptor subtypes (2). Up to seven P 2X receptor subtypes (3-9) and eight P 2Y receptor subtypes (10 -16) have been cloned from mammalian and avian species. Fuller understanding of the functions subserved by discrete receptor subtypes is hampered by the organization of native P 2X receptors into homopolymers or heteropolymers (5) and by the co-existence of P 2X and P 2Y receptors on the same cell (17). Earlier classifications based on agonist potency profiles had been confounded by the paucity of selective antagonists and radioligands (2), and by the rapid degradation of some nucleotides, mainly ATP and 2-methylthio-ATP, by ecto-nucleotidases (18), and the interconversion of adenine and uridine nucleotides by ecto-nucleoside diphosphokinases (19,20). P 2X1 is the main P 2X receptor subtype expressed in visceral and vascular smooth muscle (21), whereas P 2X2 and P 2X3 are the main receptor subtypes expressed in peripheral sensory ganglia (8,(21)(22)(23). Both P 2X1 and P 2X3 receptors have high affinity for ATP and AMP-PCP 1 and are rapidly desensitized (23, 24). P 2X2 , P 2X4 , and P 2X6 receptors are the predominant receptor s...

Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in LβT2 Cells

“…Activation of phospholipase C would be consistent with coupling to G␣ q as this G protein can activate PLC␤1 and ␤3 (26). However, activation can also be G␣ q -independent as G␤␥ can activate PLC␤2 (27).…”

Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

“…PI hydrolysis via m3 receptors was inhibited by PLC-β1 and Gα q/11 antibodies, whereas PI hydrolysis via m2 receptors was inhibited by PTx, and PLC-β3 and Gβ antibodies (Figure 1). Activation of PLC-β3 followed a well-established pattern of activation of this isoenzyme in smooth muscles by βγ subunits of G i1 , G i2 and G i3 [39][40][41][42][43]. The effectiveness of PLC-β1 and -β3 antibodies and antibodies to Gα and Gβ subunits in blocking responses in muscle cells has been characterized previously [39][40][41][42][43].…”

“…By analogy with other G i3 -(or G i1/2 -) coupled receptors (e.g. adenosine A 1 receptors [40] and purine/ pyrimidine P2Y 2 receptors [41], opioid µ, δ and κ receptors [42] and somatostatin sstr 3 receptors [43]), which stimulate PI hydrolysis and Ca 2+ mobilization and induce contraction by activating phospholipase C-β3 (PLC-β3) via Gβγ subunits, we postulated that m2 receptors would, similar to m3 receptors, be capable of initiating contraction. Unexpectedly, however, m2 receptors appeared to be inert.…”

“…Smooth-muscle cells were isolated from the circular muscle layer of rabbit intestine by sequential enzymic digestion, filtration and centrifugation as described previously [41,43]. For some experiments, the cells were placed in a culture in Dulbecco's modified Eagle's medium containing 10 % (v/v) foetal bovine serum until they attained confluence [44].…”

“…Phosphorylation of CPI-17, MYPT1 and MLC 20 was measured by Western-blot analysis using phospho-specific antibodies as described previously [25,37,[41][42][43]. Dispersed muscle cells were treated with ACh for different time periods in the presence or absence of diphenylacetoxy-N-methylpiperidine (4-DAMP), methoctramine, bisindolylmaleimide or Y27632 and solubilized on ice for 1 h in a medium containing 20 mM Tris/HCl (pH 8.0), 1 mM DTT, 100 mM NaCl, 0.5 % SDS, 0.75 % deoxycholate, 1 mM PMSF, 10 µg/ml leupeptin and 100 µg/ml aprotinin.…”